Cocarboxylase arginate and process for the production thereof

ABSTRACT

A stable cocarboxylase arginate is prepared by reacting one mole of cocarboxylase with at least one mole of arginine or a salt thereof. The cocarboxylase arginate product may be recovered from the reaction mixture by conventional techniques, and has excellent moisture stability and an enhanced pharmaceutical activity.

United States Patent [191 Kondo et al.

[ 1 Jan.30, 1973 Inventors:

Seigo Kondo, I-Iorinouchi, Suginamiku, Tokyo; Toshikazu Tabata,Kohoku-ku, Yokohama-shi, KanagaWa-ken; Jiro Horiuchi, Sayama-shi,Saitama-ken, all of Japan Assignee: Kanto Ishiseiyaku Co., Ltd.,

Saitama-ken, Japan Filed: Oct. 26, 1970 Appl. No.: 84,180

Foreign Application Priority Data Oct. 24, I969 Japan ..44/85l l4 U.S.Cl...........260/256.5 B, 260/256.6, 424/255 Int. Cl. ..C07d 99/12 Fieldof Search ..260/256.5 B, 256.6

[56] References Cited UNITED STATES PATENTS 3,432,504 3/l969Goetze-Claren ..260/256.6

FOREIGN PATENTS OR APPLICATIONS 683,178 3/l964 Canada ..260/256.5 B

Primary Examiner-Alex Mazel Assistant Examiner-R. J. Gallagher Att0rney-Oblon, Fisher & Spivak ABSTRACT A stable cocarboxylase arginate isprepared by reacting one mole of cocarboxylase with at least one mole ofarginine or a salt thereof. The cocarboxylase arginate product may berecovered from the reaction mixture by conventional techniques, and hasexcellent moisture stability and an enhanced pharmaceutical activity.

1 Claim, No Drawings COCARBOXYLASE ARGINATE AND PROCESS FOR THEPRODUCTION THEREOF BACKGROUND OF THE INVENTION 1. Field of The InventionThis invention relates to a new chemical compound and a novel processfor its preparation. More particularly, this invention relates tococarboxylase arginate and a process for the preparation thereof byreacting cocarboxylase with arginine or a salt thereof, andcrystallizing the resulting product.

2. Description Of The Prior Art Thiamine pyrophosphate is a knownco-enzyme which acts with carboxylase in the in vivo oxidativedecarboxylation of a-keto acids, such as in the decarboxylation ofpyruvic acid into acetaldehyde and carbon dioxide. As such, thiaminepyrophosphate is often referred to as cocarboxylase, although it is alsoa necessary co-enzyme for the transketolation reactions which occur inthe direct oxidative pathway of glucose metabolism.

Cocarboxylase has many clinical and experimental uses which are wellknown in the art. However, cocarboxylase is very unstable in thepresence of moisture and loses its activity not only when stored in theform of an aqueous solution, but also when stored as a powder or tabletdue to the adsorption of moisture during storage.

SUMMARY OF THE INVENTION It is a principal object of this invention toprovide a cocarboxylase arginate which is stable in the form of anaqueous solution.

Another object of this invention is to provide a cocarboxylase arginatewhich is stable in the form of a powder or tablet.

A further object of this invention is to provide a method of preparing acocarboxylase arginate which will remain stable in the presence ofmoisture.

An additional object of this invention is to provide a cocarboxylasearginate having enhanced pharmaceutical activity. 1

Yet another object of this invention is to provide a cocarboxylasearginate having wide pharmaceutical applications.

Briefly, these and other objects are attained in one aspect of thepresent invention which provides a unique cocarboxylase arginate andsimple methods for the preparation thereof. As a result of researchconducted to solve the above-mentioned and other disadvantages ofcocarboxylase, it has now been discovered that cocarboxylase arginatemay be prepared by reacting cocarboxylase with arginine or its salts,and that the carboxylase arginate thus prepared is characterized bygreatly improved moisture stability and enhanced biochemical properties.

DESCRIPTION OF AN ILLUSTRATIVE EMBODIMENT According to the presentinvention, cocarboxylase arginate is prepared by reacting cocarboxylasewith arginine or an arginine salt. Preferably a slight molar excess ofarginine will be reacted with each mole of cocarboxylase in order toobtain a good yield.

In carrying out the reaction, a solvent which will dissolve bothcocarboxylase and arginine is utilized, preferably using a minimumamount of solvent which will dissolve both the cocarboxylase and thearginine or arginine salt. Suitable solvents are aqueous solvents suchas water or a mixture of water with an organic solvent, such asmethanol, ethanol, glycol, glycerine or the like.

The reaction may be carried out at room temperature or, if desired,under gentle heating.

In order to separate and purify cocarboxylase arginate from the reactionmixture, conventional recovery methods may be utilized. One suitablemethod is to add a non-solvent for cocarboxylase arginate, such asethanol, to the reaction mixture and collect the precipitate whichcrystallizes upon cooling. The collected precipitate may be dissolved inwater, ethanol again added, and the mixture allowed to stand in a coldplace so that cocarboxylase arginate will deposit and crystallize. Othersuitable recovery methods will be apparent to those skilled in the art.

The following Example is illustrative of one suitable method by whichthe new compound of the present invention can be prepared.

EXAMPLE Two-hundred g. of arginine is dissolved in 2,000 ml. of warmwater, 493 g. of cocarboxylase is added to the solution, and the mixtureis reacted at room temperature for 30 minutes. The reaction mixture isfiltered, 4,000 ml. of ethanol is added to the filtrate, and theresulting mixture is stirred and allowed to stand in a cold place. 630g. of precipitate is deposited. The precipitate is then dissolved in 200ml. of warm water and filtered. 4,000 ml. of ethanol is added to thefiltrate, and the mixture is allowed to stand in a cold place overnightto deposit colorless crystals. The crystals are collected by filtrationand dried to obtain 500 g. (representing a 74.4 percent yield ofcocarboxylase arginate melting at 1 l0 C. with decomposition.

While the detailed chemical structure of the thus-obtained cocarboxylasearginate is not yet known, it is believed to be a single compound inwhich one mole of cocarboxylase has reacted with one mole of arginine,as established from the infra-red and ultra-violet absorption spectra.Cocarboxylase arginate melts over a range of 1 10 120C. withdecomposition, and is very stable, even in the presence of moisture.

The Table below sets forth the stability of cocarboxylase arginate ascompared with a control of cocarboxylase. Each compound was dissolvedinwater to make a separate solution of 25 mg. per l0 ml., and stabilitydata were obtained by allowing the solutions to stand at temperatures of37C., 45 C., and 60 C. for several months. Activity was measured at eachsample interval and compared with the initial activity of the freshlyprepared solutions, expressed as a per cent survival figure for eachsample.

TABLE: STABILITY DATA, PER CENT SURVIVAL Months Temperature 1 3 4 6 8 l218 A2 Cocarboxylase B: Cocarboxylase arginate In addition to the greatlyimproved stability shown above, cocarboxylase arginate has excellentpharmaceutical properties. As one example thereof, the cocarboxylaseactivity is greatly enhanced in cocarboxylase arginate, which is on theorder of ten times as effective as cocarboxylase.

Furthermore, excellent clinical therapeutic results have been obtainedwith cocarboxylase arginate, even for conditions in which cocarboxylaseand activated Vitamin B treatment had no recognizable effect, such as intreatment of the sequelae of external head wounds, whiplash injuries,certain hearing difficulties, acute or chronic hepatitis, apoplexy, andpost-operative intestinal paralysis.

It will be appreciated that while the foregoing disclosure isspecifically directed to the preparation and use of cocarboxylasearginate, it is capable of numerous modifications and alterationsthereof, with respect to the preparation of cocarboxylase arginate andits clinical use. Accordingly, numerous modifications or alterations maybe made by those skilled in the art without departing from the spiritand scope of the present invention as set forth in the appended claims.

What is claimed and desired to be secured by letters patent of theUnited States is:

l. Cocarboxylase arginate.

